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S D Ehrlich

Publications and source records attributed to S D Ehrlich.

At least 127 records · Page 7Linked to original sources
PubMed1995

Cloning and characterization of the Bacillus subtilis birA gene encoding a repressor of the biotin operon.

The Bacillus subtilis birA gene, which regulates biotin biosynthesis, has been cloned and characterized. The birA gene maps at 202 degrees on the B. subtilis chromosome and encodes a 36,200-Da protein that is 27% identical to Escherichia coli BirA protein. Three independent mutations in birA that lead to deregulation of biotin synthesis alter single amino acids in the amino-terminal end of the protein. The amino-terminal region that is affected by these three birA mutations shows sequence similarity to the helix-turn-helix DNA binding motif previously identified in E. coli BirA protein. B. subtilis BirA protein also possesses biotin-protein ligase activity, as judged by its ability to complement a conditional lethal birA mutant of E. coli.

Amino Acid Sequence↗
PubMed1995

Metabolic operons in Lactococci.

The genes for the biosynthesis of histidine, tryptophan and branched-chain amino acids (ilv for isoleucine, leucine and valine) are clustered in large operons. In addition to genes encoding the pathway enzymes, the his and the ilv operons contain 4 and 3 other genes, respectively. The functions of two of these, orf3 and aldB are regulatory. The second gene of the his operon, orf3, encodes a protein homologous to the histidyl-tRNA synthetases. It is involved in transcription attenuation upstream of the his operon. This regulation is related to a new class of attenuation mechanisms controlling the expression of most tRNA synthetase genes and a few metabolic operons in Gram-positive bacteria. Gene aldB, the penultimate gene of the ilv operon, encodes acetolactate decarboxylase. This enzyme transforms acetolactate (AL), the first intermediate of leucine and valine biosynthesis, into acetoin. AL decarboxylase is positively controlled by the availability of leucine and possibly valine in the cell. This is the key enzyme for a new class of regulatory mechanisms, a metabolic shunt which guides the AL flux towards synthesis of amino acids or a secondary metabolite.

Amino Acid Sequence↗
PubMed1995

recA gene involvement in oxidative and thermal stress in Lactococcus lactis.

The recA gene is best known for its effects on homologous recombination and DNA repair via SOS induction. There is gathering evidence that recA also affects expression of genes associated with different types of stress. We studied recA properties in Lactococcus lactis by generating a recA-disrupted mutant of MG1363 and comparing it with the wild type strain. recA appears to have an important role in cell survival upon oxygen or thermal stress, in addition to its conserved role in DNA repair. Oxygen toxicity appears to be due to the production of hydroxyl radicals via the Fenton reaction; recA would be involved in the repair of DNA damage generated by these radicals. Surprisingly, the recA strain stops growing at elevated temperature (37 degrees C). Immunological tests indicate that amounts of three heat shock proteins are reduced in the recA strain compared to the wild type strain. In contrast, the amount of heat shock regulator HflB is markedly increased, even at low temperature. HflB is known to degrade heat shock transcription factor sigma 32 in Escherichia coli. We propose that heat shock response is reduced in the recA mutant due to overproduction of HflB.

ATP-Dependent Proteases↗
PubMed1995

Specificity of insertion of Tn1545 transposon family in Lactococcus lactis subsp. lactis.

A collection of Lactococcus lactis subsp. lactis strains carrying a derivative of Tn1545 inserted in the chromosome was generated. Some 34 insertions were cloned in Escherichia coli and the flanking DNA sequences determined. Insertions are distributed non randomly and several hot spots were observed. The hot spots do not have sequence features which would distinguish them from other insertion sites, suggesting that they may have special structural properties. Insertions in open reading frames occurred at a far lower frequency than expected for random transposition and were most often located near potential terminator and promoter sequences. Different mutations in the extremities of the transposon do not affect the specificity of insertion. We suggest that target specificity is mainly due to the properties of the intergrase itself.

Amino Acid Sequence↗
PubMed1994

Active site of the replication protein of the rolling circle plasmid pC194.

Mutation analysis of the rolling circle (RC) replication initiator protein RepA of plasmid pC194 was targeted to tyrosine and acidic amino acids (glutamate and aspartate) which are well conserved among numerous related plasmids. The effect of mutations was examined by an in vivo activity test. Mutations of one tyrosine and two glutamate residues were found to greatly impair or abolish activity, without affecting affinity for the origin, as deduced from in vitro gel mobility assays. We conclude that all three amino acids have a catalytic role. Tyrosine residues were found previously in active sites of different RC plasmid Rep proteins and topoisomerases, but not in association with acidic residues, which are a hallmark of the active sites of DNA hydrolyzing enzymes, such as the exo- and endonucleases. We propose that the active site of RepA contains two different catalytic centers, corresponding to a tyrosine and a glutamate. The former may be involved in the formation of the covalent DNA-protein intermediate at the initiation step of RC replication, and the latter may catalyze the release of the protein from the intermediate at the termination step.

Amino Acid Sequence↗
PubMed1994

Effect of length and location of heterologous sequences on RecA-mediated strand exchange.

We systematically investigated the effect of heterology on RecA-mediated strand exchange between double-stranded linear and single-stranded circular DNA. Strand exchange took place through heterologies of up to 150-200 base pairs when the insertion was at the proximal (initiating) end of the duplex DNA but was completely blocked by an insert of only 22 base pairs placed at the distal end of the duplex. In the case of medial heterology created by insertion either in the duplex or the single-stranded DNA, the ability of RecA to exchange strands decreased as the heterology was shifted toward the distal end of the duplex. These results suggest that two different strand exchange mechanisms operate in the proximal and distal portions of the duplex substrate.

Bacteriophage M13↗
PubMed1994

Characterization of IS1201, an insertion sequence isolated from Lactobacillus helveticus.

IS1201, a 1387-bp insertion sequence isolated from Lactobacillus helveticus, was identified by its nucleotide (nt) sequence. It carries a single open reading frame encoding a 369-amino-acid protein, which shares homology with transposases found in a class of related IS, including ISRm3 from Rhizobium meliloti, IS256 from Staphylococcus aureus, IS6120 from Mycobacterium smegmatis, IS1081 from M. bovis, IST2 from Thiobacillus ferroxidans and IS406 from Pseudomonas cepacia. IS1201 has terminal inverted repeats of 24 bp in length and a target site duplication of 8 bp. Its copy number ranges from 3 to about 16 per L. helveticus genome. No homology was found between the nt sequence of IS1201 and those of the other bacterial IS from the same class. These results, together with previous observations [de los Reyes-Gavilán et al., Appl. Environ. Microbiol., 58 (1992) 3429-3432], confirm that IS1201 can be used as a specific DNA probe for the identification of L. helveticus strains.

Amino Acid Sequence↗
PubMed1994

Copy-choice illegitimate DNA recombination revisited.

Nearly precise excision of a transposon related to Tn10 from an Escherichia coli plasmid was used as a model to study illegitimate DNA recombination between short direct repeats. The excision was stimulated 100-1000 times by induction of plasmid single-stranded DNA synthesis and did not involve transfer of DNA from the parental to the progeny molecule. We conclude that it occurred by copy-choice DNA recombination, and propose that other events of recombination between short direct repeats might be a result of the same process.

Base Sequence↗
PubMed1994

The promiscuous plasmids pIP501 and pAM beta 1 from gram-positive bacteria encode complementary resolution functions.

pIP501 and pAM beta 1 are promiscuous plasmids from gram-positive bacteria which carry structurally and genetically related replication functions (L. Jannière, A. Gruss and S.D. Ehrlich, 1993, in "Bacillus subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology, and Molecular Genetics (A.L. Sonenshein, J.A. Hoch, and R. Losick, Eds.), pp. 625-644). We report here the sequence of a approximately 1 kb region located downstream of the pIP501 minimal replicon. This region is 82% homologous to the corresponding region of pAM beta 1 and encodes a resolution system which complements that of pAM beta 1. Additionally, we present evidence that the structural similarity between the two plasmids extends for further approximately 2 kb, through a region which may encode a topoisomerase protein in pAM beta 1.

Amino Acid Sequence↗
PubMed1994

Mosaic structure of large regions of the Lactococcus lactis subsp. cremoris chromosome.

Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris are closely related phenotypically and genetically. Here we report that certain regions of their chromosomes diverge considerably more than others. Conserved regions differ by less than 20%, whilst variable regions differ by more than 60%. This mosaic structure may have arisen by horizontal gene transfer from distantly related bacteria since in a particular region of the L. lactis subsp. cremoris chromosome the G+C content and the codon bias are not typical for lactococci. Such an exchange, which conserves the function of the gene and cannot be achieved under selective pressure, may be of considerable importance in the evolution of bacteria.

Amino Acid Sequence↗
PubMed1994

Sequence and organization of the lactococcal prolate-headed bIL67 phage genome.

bIL67 is a broad-host-range prolate-headed phage that is active against Lactococcus cells. The complete phage genome sequence of 22195 bp was established. Thirty-seven open reading frames (ORFs) organized in two clusters were identified. Functions were assigned to the putative products of six of the ORFs on the basis of comparison of the deduced amino acid sequences to known proteins, analysis of structural features of the proteins and search for conserved motifs. These were a DNA polymerase, a protein involved in recombination, a lysin, a terminase subunit, a structural protein and a holin.

Amino Acid Sequence↗
PubMed1994

Frequency of deletion formation decreases exponentially with distance between short direct repeats.

The effect of distance between 18 bp direct repeats on deletion formation has been examined in Bacillus subtilis. The deletion frequency decreased exponentially by more than 1000-fold as the distance increased from 33 to 2313 bp. This decrease occurred in two distinct phases, which may be determined by DNA-duplex flexibility. A similar relationship between deletion formation and distance was observed in a theta-replicating plasmid and in the chromosome, indicating that this relationship might have a general validity.

Bacillus subtilis↗
PubMed1994

The pAM beta 1 CopF repressor regulates plasmid copy number by controlling transcription of the repE gene.

pAM beta 1 is a low-copy-number, promiscuous plasmid from Gram-positive bacteria that replicates by a unidirectional theta-type mode. Its replication is initiated by an original mechanism, involving the positive rate-limiting RepE protein. Here we show that the pAM beta 1-encoded CopF protein is involved in negative regulation of the plasmid copy number. CopF represses approximately 10-fold the transcription initiated at the promoter of the repE gene and binds to a 31 bp segment which is located immediately upstream of the -35 box of the repE promoter. We propose that CopF inhibits initiation of transcription at the repE promoter by binding to its operator.

Bacillus subtilis↗
PubMed1994

Flanking sequences affect replication arrest at the Escherichia coli terminator TerB in vivo.

We have analyzed the effect of flanking sequences on Tus-induced replication arrest. pBR322 plasmid derivatives which carry the Escherichia coli replication terminator TerB at different locations were used. Efficiency of the replication arrest was estimated from the plasmid copy number and transformation frequency of tus+ cells. We found that flanking sequences do affect replication arrest efficiency, a weak arrest being correlated with the presence of an AT-rich region which is replicated just before TerB. Some sequences located after the replication terminator can also affect replication termination. We propose that the AT-rich regions might impair binding of the Tus protein to the TerB sequence or facilitate helicase-induced unwinding of DNA and Tus displacement from the TerB site.

Bacterial Proteins↗
PubMed1994

Direct screening of recombinants in gram-positive bacteria using the secreted staphylococcal nuclease as a reporter.

A system for direct screening of recombinant clones in Lactococcus lactis, based on secretion of the staphylococcal nuclease (SNase) in the organism, was developed. The nuc gene (encoding SNase) was cloned on both rolling-circle and theta-replicating plasmids. L. lactis strains containing these nuc+ plasmids secrete SNase and are readily detectable by a simple plate test. A multicloning site (MCS) was introduced just after the cleavage site between leader peptide and the mature SNase, without affecting nuclease activity. Cloning foreign DNA fragments into any site of the MCS interrupts nuc and thus results in nuc mutant clones which are easily distinguished fron nuc+ clones on plates. The utility of this system for L. lactis was demonstrated by cloning an antibiotic resistance marker and Escherichia coli chromosomal DNA fragments into the MCS of the nucMCS cassette. Both cloning vectors containing the nucMCS cassette were also introduced into Streptococcus salivarius subsp. thermophilus, in which direct screening of nuc mutant recombinant clones was also achieved. The potential uses of nuc as a secretion reporter system are discussed.

Base Sequence↗
PubMed1994

BglR protein, which belongs to the BglG family of transcriptional antiterminators, is involved in beta-glucoside utilization in Lactococcus lactis.

A fragment of the Lactococcus lactis chromosome containing an open reading frame of 265 codons, denoted bglR, has been characterized. The polypeptide encoded by bglR shares 36 to 30% sequence identity with a family of regulatory proteins including ArbG from Erwinia chrysanthemi, BglG from Escherichia coli, and SacT and SacY from Bacillus subtilis. These regulatory proteins are involved in positive control of the utilization of different sugars by transcription antitermination. For some of these regulatory proteins it has been demonstrated that antitermination is exerted by binding to a conserved RNA sequence, partially overlapping the transcription terminator and thus preventing transcription termination. Upstream of bglR, we identified a transcription terminator whose 5' end was overlapped by a 32-bp sequence, highly homologous to the RNA-binding site that is conserved in other regulatory systems. Constitutive expression of bglR in E. coli increased the expression of a bglG::lacZ transcriptional fusion. The fact that that the expression of BglG is autoregulated in E. coli suggests that BglG and BglR are functionally equivalent. In L. lactis, we observed that (i) the expression of a bglR::lacZ fusion is increased by beta-glucoside sugars, (ii) disruption of bglR impairs growth on some beta-glucosides, and (iii) the expression of bglR is positively autoregulated. Because of these structural and functional similarities between BglR and the transcription antiterminators of the BglG family, we propose that BglR may be the lactococcal counterpart of the E. coli BglG regulator of beta-glucoside utilization.

Amino Acid Sequence↗
PubMed1993

Mechanisms of illegitimate recombination.

Illegitimate recombination, which is one of the major causes of genome rearrangements, can occur in a number of ways. These might involve enzymes which cut and join DNA or enzymes which replicate DNA, as illustrated by two examples: (i) formation of deletions at the replication origin (ori) of an Escherichia coli bacteriophage, M13; and (ii) excision of E. coli transposon Tn10. It is proposed that a common theme to various ways by which illegitimate recombination can occur might be the capacity to create ends in the DNA molecule and to make the ends meet.

Bacteriophage M13↗